BioMed Central Page 1 of 9 (page number not for citation purposes) Virology Journal Open Access Research Identification of a truncated nucleoprotein in avian metapneumovirus-infected cells encoded by a second AUG, in-frame to the full-length gene Rene Alvarez 1,2 and Bruce S Seal* 1,3 Address: 1 Southeast Poultry Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Athens, GA 30605, USA, 2 Present address: Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30605, USA and 3 Poultry Microbiological Safety Research Unit, ARS, USDA, 950 College Station Rd., Athens, GA 30605, USA Email: Rene Alvarez - ralvarez@vet.uga.edu; Bruce S Seal* - bseal@saa.ars.usda.gov * Corresponding author Abstract Background: Avian metapneumoviruses (aMPV) cause an upper respiratory disease with low mortality, but high morbidity primarily in commercial turkeys. There are three types of aMPV (A, B, C) of which the C type is found only in the United States. Viruses related to aMPV include human, bovine, ovine, and caprine respiratory syncytial viruses and pneumonia virus of mice, as well as the recently identified human metapneumovirus (hMPV). The aMPV and hMPV have become the type viruses of a new genus within the Metapneumovirus. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. Based on predicted antigenicity of consensus protein sequences, five aMPV-specific N peptides were synthesized for development of peptide-antigens and antisera. Results: The presence of two aMPV nucleoprotein (N) gene encoded polypeptides was detected in aMPV/C/US/Co and aMPV/A/UK/3b infected Vero cells. Nucleoprotein 1 (N1) encoded from the first open reading frame (ORF) was predicted to be 394 amino acids in length for aMPV/C/US/Co and 391 amino acids in length for aMPV/A/UK/3b with approximate molecular weights of 43.3 kilodaltons and 42.7 kilodaltons, respectively. Nucleoprotein 2 (N2) was hypothesized to be encoded by a second downstream ORF in-frame with ORF1 and encoded a protein predicted to contain 328 amino acids for aMPV/C/US/Co or 259 amino acids for aMPV/A/UK/3b with approximate molecular weights of 36 kilodaltons and 28.3 kilodaltons, respectively. Peptide antibodies to the N-terminal and C-terminal portions of the aMPV N protein confirmed presence of these products in both aMPV/C/US/Co- and aMPV/A/UK/3b-infected Vero cells. N1 and N2 for aMPV/C/US/Co ORFs were molecularly cloned and expressed in Vero cells utilizing eukaryotic expression vectors to confirm identity of the aMPV encoded proteins. Conclusion: This is the first reported identification of potential, accessory in-frame N2 ORF gene products among members of the Paramyxoviridae. Genomic sequence analyses of related members of the Pneumovirinae other than aMPV, including human respiratory syncytial virus and bovine respiratory syncytial virus demonstrated the presence of this second potential ORF among these agents. Published: 12 April 2005 Virology Journal 2005, 2:31 doi:10.1186/1743-422X-2-31 Received: 04 April 2005 Accepted: 12 April 2005 This article is available from: http://www.virologyj.com/content/2/1/31 © 2005 Alvarez and Seal; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Virology Journal 2005, 2:31 http://www.virologyj.com/content/2/1/31 Page 2 of 9 (page number not for citation purposes) Background Avian metapneumovirus (aMPV) causes turkey rhinotra- cheitis (TRT) and is associated with swollen head syn- drome (SHS) of chickens that is usually accompanied by secondary bacterial infections which can increase morbid- ity and induce mortality. Avian metpnuemovirus was first reported in South Africa during the early 1970s and was subsequently isolated in Europe, Israel and Asia [1,2]. During 1997, mortality due to aMPV infections among commercial turkeys in the U.S. ranged from zero, to 30% when accompanied by bacterial infections, with condem- nations due to air sacculitis. This was the first reported outbreak of aMPV infections in the U.S. which was previ- ously considered exotic to North America. The virus caus- ing disease was designated a new aMPV type C genetically different from European counterparts [3-5] and was sub- sequently demonstrated to be most closely related to human metapneumovirus (hMPV) from diverse geo- graphic locations [6,7]. Infections among commercial tur- keys with aMPV/C continue in the north-central U.S. resulting in substantial economic loss to the poultry industry [6,8,9]. Pneumoviruses are members of the family Paramyxoviri- dae that contain a nonsegmented, negative-sense RNA genome of approximately 15 kb in length. Viruses related to aMPV include human, bovine, ovine and caprine respi- ratory syncytial viruses and pneumonia virus of mice [10], as well as the recently identified hMPV [11]. Although genome length is similar, pneumoviruses generally encode ten genes, compared to six or seven in other para- myxoviruses. These include the nonstructural proteins (NS1 and NS2), nucleoprotein (N), phosphoprotein (P), matrix protein (M), small hydrophobic protein (SH), sur- face glycoprotein (G), fusion protein (F), second matrix protein (M2) and a viral RNA-dependent RNA polymerase (L). The pneumoviruses have an F protein that promotes cell fusion, but these viruses do not hemagglutinate, nor do they have neuraminidase activity in their G attachment protein. This is an important distinguishing characteristic from the other paramyxoviruses [10]. Because of a limited genome size, many non-segmented RNA viruses, including the pneumoviruses, have devised mechanism to increase protein coding capacities. This may occur at two levels: 1) transcriptional mRNA process- ing or modification [12-14] or 2) translational, in which proteins may be produced from alternative open reading frames (ORFs) or from translational initiation at non- AUG or downstream AUG codons [15-17]. Among the pneumoviruses, secondary coding usage has only been documented for the M2 gene, which encodes two pro- teins. The M2-1, a transcription antitermination factor, is required for processive RNA synthesis and transcription read-through at gene junctions. The M2-2 is involved with the shift between viral RNA transcription and replication [18]. In this report, we present evidence for utilization of a secondary open reading frame, within the N gene encod- ing a truncated nucleoprotein (N2) among aMPV/C/Co and aMPV/A/UK/3b infected cells. Results Avian metapneumovirus N gene possess several putative AUG start sites The aMPV/C/US/Co nucleoprotein is encoded by the N gene with a predicted molecular weight of 42–45 kD [7,19]. The N gene ranges from 1191 to 1206 nucleotides in length [6,19], with the first AUG at nucleotide position 14 (Fig. 1) in all three subtypes (A, B, and C). The aMPV/ C/US/Co N gene has additional putative start sites at nucleotide positions 212, 350, 416, 758, 785, 827, 896, and 1022 with "true" Kozak sequences [20] at nucleotide positions 413 (ACCAUG G) and 893 (GAGAUGG), with predicted translation products of 28.5 kD and 10.78 kD, respectively. The aMPV/A/UK/3b N gene has additional putative start sites at nucleotide positions 161, 212, 293, 410, 413, 605, 722, 749, 749, 776, 818, 887, and 1013 with "true" Kozak sequences [20] at nucleotide positions 602 (AGGAUG G), 719 (AGGAUGG), and 884 (AAAAUG G), with predicted translation products of 21.26 kD, 16.73 kD, and 10.54 kD, respectively. Avian metapneumovirus-infected cells produce two proteins (N1 and N2) encoded by two open-reading frames within the N gene Five peptides within the aMPV N gene (Fig. 2) were uti- lized to generate affinity-purified rabbit peptide antibod- ies. This approach was exploited to determine if any of the alternative start sites of the aMPV N gene were utilized during an active cell infection. aMPV/N-peptide antibody directed against aMPV/C/US/Co N protein amino acids 10–29 (DLSYKHAILKESQYTIKRDV) with only 3 changes in both aMPV types A and B at amino acid positions 12 (S to E), 19 (K to D) and 26 (K to R) reacted with all three full length nucleoproteins by western blot (Fig. 3A, Lanes 3, 4, and 5), but did not react with any proteins in unin- fected Vero cells (Fig. 3A, Lane 2). All three virus nucleo- proteins were between 42–45 kD based on SDS-PAGE/ western blot analysis (Fig. 3A). We then tested the aMPV/ C-N2 peptide antibody directed against amino acids 128– 148 in the mid-portion of the of the aMPV/C/US/Co iso- late (Fig. 2) by western blot which would recognize any downstream translation products encoded by the N gene and utilization of any secondary start sites. Western blot analysis revealed two putative N gene products in aMPV/ C/US/Co-infected Vero cells, the first, the full-length nucleoprotein with a molecular weight of approximately 43 kD (Fig. 3B, Lane 3) and the second, a smaller protein of approximately 35–36 kD (Fig. 3B, Lane 3). The peptide antibody to amino acids 303 to 393 (aMPV/C-N4) Virology Journal 2005, 2:31 http://www.virologyj.com/content/2/1/31 Page 3 of 9 (page number not for citation purposes) synthesized to be reactive to the C-terminal N protein from aMPV/C also recognized two proteins as in Fig. 3B, Lane 3 (data not shown). To evaluate whether the utilization of alternative start sites was unique to members of the aMPV type C group, or whether this also occurred in other aMPV types, we uti- lized aMPV/A-N3 and aMPV/A-N5 peptide antibodies (anti-aMPV/Type A, N protein, amino acids 126–145 and 380–390, respectively). Unlike aMPV/C-N2 peptide anti- body, aMPV/A-N3-peptide antibody (amino acids 126– 145) reacted to only a full length nucleoprotein (Fig. 3C, lane 3) similar to the aMPV/N-peptide antibody (Fig. 3C, lane 2), while aMPV/A-N5-peptide antibody (amino acids 380–390) reacted with both the full length nucleoprotein of approximately 41–43 kD (Fig. 3C, lane 4) and a smaller protein of approximately 28–30 kD (Fig. 3C, lane 4). Finally, all aMPV type-specific antibodies were not cross active with other metapneumoviruses (data not shown). Expression of the N1 and N2 ORF of avian metapneumovirus type C/Colorado in eukaryotic cells Sequence analysis of the aMPV/C/US/Co and aMPV/A/ UK/3b N gene nucleotide sequences revealed that down- stream of the first AUG (position 14) were multiple puta- tive start sites as described above (Fig. 1). We therefore utilized sequence analysis software to analyze the N gene putative open reading frames and the predicted transla- tion products from each putative start site for products that would result in proteins of approximate size as the smaller reactive band that was detected by western blot (Fig. 3B, lane 3 and Fig. 3C, lane 4). Two predicted pro- teins in the aMPV/C/US/Co sequences corresponding to a predicted molecular weight of approximately 31.12 kD Alignment of avian metapneumovirus type A and C nucleoprotein genes demonstrating presence of multiple start sitesFigure 1 Alignment of avian metapneumovirus type A and C nucleoprotein genes demonstrating presence of multiple start sites. Under- lined sequences denote hypothesized alternative in-frame start sites and the stop codon. Primer sequences utilized for cDNA synthesis of nucleoprotein genes are also illustrated. N1 ______________________________ aMPV/C/US/Co GGGACAAGTG AAAATGTCTC TTCAGGGGAT TCAGCTTAGT GACTTGTCCT ATAAGCATGC AATCCTTAAA GAATCACAGT ACACAATCAA 90 aMPV/A/UK/3b GGGACAAGTC AAAATGTCTC TTGAAAGTAT TAGACTCAGT GACTTGGAGT ACAAACATGC AATTCTTGAA GACTCTCAGT ATACAATTAG 90 aMPV/C/US/Co AAGAGATGTG GGGACAACCA CAGCTGTCAC TCCGTCTTCT CTGCAGAGGG AAGTGTCACT CTTATGTGGA GAGATACTGT ATGCCAAGCA 180 aMPV/A/UK/3b AAGGGATGTT GGTGCTACCA CTGCGATCAC ACCTTCCGAA CTGCAGCCGC AAGTATCCAC ATTATGCGGT ATGGTGTTGT TTGCAAAACA 180 N212 ____________________ aMPV/C/US/Co CACAGATTAC TCACATGCAG CTGAAGTAGG AATGCAGTAC GTGAGCACCA CACTGGGAGC AGAGCGTACA CAGCAGATAC TAAAGAACTC 270 aMPV/A/UK/3b CACCGACTAT GAGCCTGCAG CAGAGGTAGG CATGCAGTAC ATTAGTACTG CTCTAGGAGC TGATAGAACT CAACAAATAC TGAAAAATTC 270 aMPV/C/US/Co AGGTAGTGAG GTGCAGGCAG TATTGACCAA GACA TACT CTCTTGG-GA AGGGCAAAAA CAGCAAAGGG GAGGAGTTGC AAATGTTAGA 357 aMPV/A/UK/3b CGGTAGTGAA GTACAGGGTG TTATGACCAA GATTGTTACA CTTTCGGCAG AGGGTTCTGT CAGAAAGCGA GAGGTGCT AAACATTCAC 358 aMPV/C/US/Co CATACATGGG GTTGAAAGAA GT TGG-AT TGAAGAAGTT GACAAAGAGG CAAGGAAAAC CATGGCCTCA GCTACAAAGG ACAACTCAGG 444 aMPV/A/UK/3b GATGTA-GGT GTTGGGTGGG CTGATGATGT CGAAAGGACT ACAAGAGAAG CAATGGGAGC AATGG TTA GGGAAAAAGT GCAACTCA 443 aMPV/C/US/Co ACCAATACCA CAAAATCAAA GACCATCATC CCCGGATGCT CCTATCATAC TACTCTGCAT AGGAGCATTA ATCTTCACGA AGCTGGCATC 534 aMPV/A/UK/3b CAA -AGAATCAAA AGCCGTCTGC CTTGGATGCT CCCGTTATTC TATTATGCAT TGGTGCCCTC ATTTTCACCA AGTTGGCCTC 525 aMPV/C/US/Co AACAATCGAA GTTGGGCTGG AGACAGCTGT TAGAAGGGCA AACCGTGTGC TGAATGATGC ATTGAAAAGG TTCCCAAGGA TTGACATCCC 624 aMPV/A/UK/3b AACTGTTGAA GTAGGCCTTG AAACTGCTAT CCGGCGTGCC TCAAGGGTAT TAAGCGATGC CATATCACGG TACCCCAGGA TGGACATACC 615 aMPV/C/US/Co CAAAATTGCG AGGTCCTTTT ATGATCTGTT TGAGCAGAAA GTTTACTACA GGAGCTTGTT TATAGAGTAT GGCAAAGCCC TTGGGTCTTC 714 aMPV/A/UK/3b AAGGATTGCC AAATCATTCT TTGAATTGTT TGAGAAGAAG GTGTATTACA GAAATCTATT TATTGAATAC GGTAAGGCAC TCGGAAGTAC 705 aMPV/C/US/Co TTCCACAGGA AGCAAGGCAG AAAGCCTGTT TGTGAATATT TTCATGCAAG CTTATGGTGC AGGTCAGACA ATGCTAAGAT GGGGGGTAAT 804 aMPV/A/UK/3b ATCCACCGGA AGCAGGATGG AGAGCCTGTT TGTGAATATT TTTATGCAAG CTTATGGGGC AGGGCAAACA ATGCTGCGCT GGGGTGTCAT 795 aMPV/C/US/Co TGCCAGATCA TCCAACAATA TAATGTTGGG CCATGTCTCC GTACAAGCAG AACTCAAACA GGTTACGGAG GTATATGATC TAGTTAGAGA 894 aMPV/A/UK/3b TGCACGATCC TCCAACAATA TAATGTTGGG CCATGTATCT GTCCAAGCTG AGTTGAGGCA AGTATCTGAG GTCTATGACC TAGTGAGGAA 885 aMPV/C/US/Co GATGGGCCCT GAGTCAGGTC TTCTTCACCT GAGGCAAAAC CCTAAGGCAG GGTTGTTGTC ACTTGCCAAT TGTCCCAATT TTGCAAGTGT 984 aMPV/A/UK/3b AATGGGACCT GAGTCAGGGT TACTACACTT ACGCCAGAGT CCCAAAGCGG GTCTTTTATC ATTGACCAAC TGTCCCAATT TTGCCAGTGT 975 aMPV/C/US/Co GGTGCTAGGG AATGCCTCAG GATTGGGGAT ACTTGGTATG TACAGAGGAA GAGTACCAAA TACAGAGCTA TTTGCCGCAG CAGAAAGCTA 1074 aMPV/A/UK/3b TGTCCTCGGG AACGCCGCCG GGCTTGGTAT TATAGGCATG TACAAAGGTC GAGCCCCCAA CCTTGAGCTG TTTGCTGCTG CTGAAAGTTA 1065 aMPV/C/US/Co TGCAAGAAGC CTAAAAGAAA GCAATAAGAT AAATTTCTCA TCTCTTGGTC TGACAGAAGA GGAAAAAGAA GCTGCTGAGA ACTTTCTCAA 1164 aMPV/A/UK/3b TGCACGGACA TTGAGAGAGA ACAACAAGAT CAACCTAGCG GCCTTAGGGC TCACTGATGA TGAGAGGGAA GCAGC-AACA TCCTACCTAG 1154 _____________________________________ N1185c aMPV/C/US/Co CATAA-ATGA -GGAAGGCCA GAATGATTAT GAGTAATTAA AAAA 1206 aMPV/A/UK/3b GGGGAGATGA TGAGAGATCA TCCAAATT-T GAGTAATTAA AAAA 1197 Virology Journal 2005, 2:31 http://www.virologyj.com/content/2/1/31 Page 4 of 9 (page number not for citation purposes) (third AUG) and another at 28.5 kD (fourth AUG) were detected in the N gene sequence. Since SDS-PAGE analysis is not necessarily an accurate measurement of molecular size, both starts sites could result in a protein observed at approximately 35–36 kD by SDS-PAGE, and therefore either site could result in the second ORF product. We therefore used two primer sets N1/N1189C and N212/N1189C which spans either the full length of ORF1 or the ORF2 and any down stream putative ORFs of aMPV/C/US/Co, respectively (Fig. 2) to amplify both ORFs by RT-PCR. Both ORFs were amplified and cloned into a eukaryotic expression vector. Western blot analysis of the Vero cell expressed N1 and N2 ORFs revealed one reactive band in the pCR3.1-N1ORF trans- fected Vero cells with the aMPV/N antibody (Fig. 4, lane 4) corresponding to the full length nucleoprotein of aMPV, similar to that observed in aMPV-infected Vero cells (Fig. 4, lane 3). This protein was not visualized in the pCR3.1-N2ORF transfected Vero cells (Fig. 4, lane 5), as was expected since the N212 primer is downstream of the peptide (aMPV/N, amino acids, 10–29) utilized to synthe- size aMPV/N peptide antibody. However, when the aMPV/C-N2 (peptide antibody directed to amino acids 383–393 of aMPV/C N protein) was used for western blot analysis, two proteins were reactive in the pCR3.1-N1ORF Vero cells, the first at approximately 43 kD (Fig. 4, lane 8), similar to that observed in aMPV-infected Vero cells (Fig. 4, Lane 7) and the second, a protein of approximately 35 kD (Fig. 4, Lane 8), slightly smaller than the N2 ORF pro- tein in aMPV-infected Vero cells (Fig. 4, Lane 7). Western blot analysis of the pCR3.1-N2ORF induced Vero cells demonstrated one reactive band of approximately 35 kD (Fig. 4, Lane 9), similar to the smaller reactive band in the pCR3.1-N1ORF transfected Vero cells. The full-length nucleoprotein, as expected was not present in the pCR3.1- N2ORF transfected Vero cells, since the N212 primer is downstream of the first AUG start site (position 14). Discussion The utilization of alternative open reading frames for the expansion of genetic information in negative-stranded RNA viruses has been well documented [10,16,17,21,22]. There are, however, various mechanisms for accessing this genetic information. The phosphoprotein of measles virus encodes a single mRNA, which is read in two Relative position of peptides within the avian metapneumovirus nucleoproteins utilized for generation of affinity purified poly-clonal antibodiesFigure 2 Relative position of peptides within the avian metapneumovirus nucleoproteins utilized for generation of affinity purified poly- clonal antibodies. aMPV/C Nucleoprotein (N) NH2- COOH 14-1196 350-1196 ORF 1 ORF 2 Peptide 1 – anti-aMPV Peptide 2 – anti-aMPV/C Peptide 3 – anti-aMPV/A Peptide 4 – anti-aMPV/C Peptide 5 – anti-aMPV/A N1 ORF N2 ORF 43.3 kD 31 kD Virology Journal 2005, 2:31 http://www.virologyj.com/content/2/1/31 Page 5 of 9 (page number not for citation purposes) independently initiated overlapping reading frames [17], while transcripts of influenza virus gene segments 7 and 8 are spliced within the nucleus for production of two dif- ferent sizes of mRNAs sharing the same 5'-proximal AUG initial codon [16]. The P gene of Sendai virus is reported to be transcribed into two polycistronic mRNAs, P/C and V/C, which are translated to synthesis the P, C, C', Y1, and Y2 proteins from independent start sites in two overlap- ping reading frames [23-25]. Within the Paramyxoviridae, Newcastle disease virus pos- sesses a polycistronic phosphoprotein (P) gene. Transcrip- tional modification of the NDV P gene mRNA allows for potential expression of two smaller putative proteins, des- ignated V and W [12], that appears to be a result of polymerase stuttering at the editing site sequences [13,14], leading to the insertion of non-template G nucle- otides within the P gene [12]. Consequently, during trans- lation there is a frame shift resulting in production of the V or W protein, dependent on the number of G nucle- otides inserted [12]. It was previously suggested that NDV [26] potentially utilized an alternative in-frame AUG start site for expression of an accessory protein similar to the Sendai virus X protein [21] that was recently demon- strated to not be utilized during infection of cells in cul- ture [27]. Pneumonia virus of mice, human and bovine respiratory syncytial viruses, and avian metapneumovirus also pos- sess polycistronic gene(s) [28-30]. The M2 gene of all the pneumoviruses contains two partially overlapping open reading frames, with the 5'-proximal open reading frame favored for utilization by the criteria of location and sequence of its start site [28,29]. The P gene of the pneu- monia virus of mice is the only known polycistronic pho- phoprotein gene in the pneumoviruses, and utilizes internal initiation of in-frame AUG initiation codons to generate up to four additional carboxy co-terminal prod- ucts [30]. In this present study, we demonstrated that the nucleo- protein gene of the avian metapneumovirus subtypes A and C are putatively polycistronic. This may occur by uti- lization of a second in-frame initiation site (AUG) for the generation of a truncated nucleoprotein present among infected Vero cells. Sequence analysis demonstrated the presence of multiple putative initiation (AUG) start sites along the N gene, however only one alternative start site at nucleotide positions 212 and 410 for APV/C and APV/ A, respectively appear to be utilized to transcribe the N2 protein seen in infected cells. The N protein of Pneumoviruses ranges in size from 42– 45 kD, based on SDS-PAGE relative mobility, and is highly conserved among metapneumoviruses [7]. The N Detection of avian metapneumovirus (aMPV) nucleoprotein gene products among infected cells utilizing affinity purified peptide antibodiesFigure 3 Detection of avian metapneumovirus (aMPV) nucleoprotein gene products among infected cells utilizing affinity purified peptide antibodies. A. Antibody reacted against an N-termi- nal portion of the nucleoprotein designed to detect all aMPV serotypes N1. Lane 1: molecular size markers; Lane 2: unin- fected cell proteins; Lane 3: aMPV/A infected cell proteins; Lane 4: aMPV/B infected cell proteins; Lane 5: aMPV/C infected cell proteins. B. Antibody detection of a C-terminal portion of the aMPV/C nucleoprotein. Lane 1: uninfected cell proteins; Lane 2: aMPV/C infected cell proteins reacted with N1 peptide antibodies; Lane 3: aMPV/C infected cells reacted with aMPV/C-specific N2 peptide antibodies. C. Antibody detection of a C-terminal portion of the aMPV/A nucleopro- tein. Lane 1: uninfected cell proteins; Lane 2: aMPV/A infected cell proteins reacted with N1 peptide antibodies; Lane 3: aMPV/A infected cell proteins reacted with N3 pep- tide antibodies; Lane 4: aMPV/A infected cells reacted with N5 peptide antibodies. A B C Virology Journal 2005, 2:31 http://www.virologyj.com/content/2/1/31 Page 6 of 9 (page number not for citation purposes) protein, which protects the RNA genome from ribonucle- ases, is associated with other viral proteins (P, M2, and L), which together form the transcription complex. The nucleocaspid is the template for transcription and replica- tion; the RNA genome by itself cannot fulfill the role of template. Pneumovirus infection in cells results in the accumulation of the N protein in cytoplasmic inclusion bodies that can be visualized by immunofluorescence [31] or immunohistochemistry [7] as relatively large dots that are usually close to the nucleus of infected cells. Mapping of several paramyxovirus N proteins, including Sendai and measles virus, indicated that the N protein has two major domains; the amino terminal domain appears to be required for nucleoprotein formation, containing the domains necessary for RNA binding and N-N interac- tions; while the carboxy-domain interacts with the phos- phoprotein (P), particularly when it is part of the polymerase complex [32,33]. In bovine respiratory syncy- tial virus (bRSV), removal of the C-terminal 32 amino acids of the N protein inhibits the interactions with the P protein, whereas the removal of 32 amino acids from the N-terminus has a minimal effect [32]. However, almost all of the N from amino acids 2–391 is required to support bRSV minigenome RNA synthesis [34]. The truncated N2 protein encompasses 328 amino acids (250 for aMPV/ Type A) of the carboxy terminus of the full-length N protein, suggesting that N2 may not be involved in the polymerase complex. However the domains responsible for RNA binding of N-N and N-P binding remain intact, suggesting that N2 may play an alternative role in cells during viral infection. Expression of N1 and N2 open reading frames of avian metapneumovirus type C in transfected eukaryotic cells by an expres-sion vectorFigure 4 Expression of N1 and N2 open reading frames of avian metapneumovirus type C in transfected eukaryotic cells by an expres- sion vector. Lane 1: molecular size markers; Lane 2: uninfected control cells; Lane 3. aMPV/C infected cells reacted with anti- bodies to peptide N1. Lane 4: Cells transformed with aMPV/C-N gene complete ORF reacted with antibodies to peptide N1. Lane 5: Cells transformed with expression plasmid with truncated N2ORF reacted to antibodies to peptide N1; Lane 6: unin- fected control cells; Lane 7: aMPV/C infected cells reacted to antibodies to peptide N4. Lane 8: Cells transformed with aMPV/ C-N gene complete ORF reacted with antibodies to peptide N2. Lane 9: Cells transformed with expression plasmid with trun- cated N2ORF reacted to antibodies to peptide N2. Virology Journal 2005, 2:31 http://www.virologyj.com/content/2/1/31 Page 7 of 9 (page number not for citation purposes) Methods Cells and viruses Vero cells were maintained as monolayer cultures in min- imal essential media (MEM) supplemented to contain 8 % fetal bovine serum with 100 units/ml penicillin G, 0.025 µg/ml amphotericin B, and 100 units/ml strepto- mycin. The aMPV/C/US/Co and aMPV/A/UK/3b isolates were obtained from the National Veterinary Services Lab- oratory (NVSL, APHIS, USDA, Ames, Iowa). Viruses were propagated on 95% confluent Vero cell monolayers in MEM supplemented to contain 2% FBS and antibiotics as described previously [3]. Cells were infected at multiplic- ity of infection of 10 (moi = 10), and virus was adsorbed for 1 hour at 37°C. Media was added and cells were incu- bated at 37°C, 5% CO 2 for 72 hours or until 90% cyto- pathic effect was observed by light microscopy. Cells were scraped and harvested by centrifugation at 8000 × g. Computer analyses, peptide synthesis and antibody production The nucleoprotein (N) gene sequences of aMPV serotypes A, B, and C (Genbank accession numbers: AAC55065, AAG42499, and AAF05909) were analyzed in the GeneWorks (Intelligentics, Mountain View, CA) and Mac Vector (Accelrys, San Diego, CA) computer analysis pro- grams to determine hydrophilicity, antigenicity, and iden- tity of the deduced amino acid sequences. The sequences were aligned for maximum similarity, and a consensus sequence was determined using the most prevalent amino acid for each residue. Five peptides with sequences: 1) aMPV/N: DLSYKHAILKESQYTIKRDV; 2) aMPV/C-N2: DKEARKTMASATKDNSGPIPQ; 3) aMPV/A-N3: ERT- TREAMGAMVREKVQLTK; 4) aMPV/C-N4: LNINEEGQNDY; and 5) aMPV/A-N5: LGGDDERSSKF were chosen based on antigenicity and hydrophilicity to be utilized for generation of aMPV peptide-based antibod- ies. Peptides were synthesized by Research Genetics (Huntsville, AL) according to the manufacturer's protocol. Briefly, rabbit aMPV/N peptide antibodies were produced by Research Genetics (Huntsville, AL) according to manu- facturer's protocol. Two rabbits were injected with 0.1 mg of KLH-conjugated peptide emulsified with Freud's com- plete adjuvant and injected into four subcutaneous (SQ) sites on day 1. On days 14, 42, and 56 rabbits were injected again (boosters) with 0.1 mg of KLH-conjugated peptide emulsified with Freud's complete adjuvant [35]. Sera were collected at days 0, 28, 56 and 70. Rabbit pre- immune sera were used as negative controls for rabbit assays. SDS-PAGE and Western blot assay Protein concentration of the supernatant fraction from infected cells was measured for protein concentration by Bradford's reagent (Bioworld, Dublin, OH) at 595 nm. Infected supernatants were denatured in Laemmli's sam- ple buffer (BioRad, Hercules, CA) and boiled for 5 min. Denatured polypeptides (6 µg protein/lane) were sepa- rated in a sodium dodecyl sulfate 4–20% polyacrylamide Criterion (Biorad, Hercules, CA) gel gradient by electro- phoresis (SDS-PAGE) at 120 V for 2 hours [36]. Polypep- tides were transferred to nitrocellulose by applying a constant voltage of 10 V for 1 hour on a Biorad (Hercules, CA) Trans-Blot SD Semi-Dry Transfer cell [37]. Blots were blocked with BLOTTO (20% dry milk in PBS) overnight at 4°C or for 1 hour at 37° and washed 3 X in phosphate buffered saline (PBS). Affinity purified rabbit anti-peptide antibody (diluted 1:100) was used as the source of the pri- mary antibodies and incubated for 1 hour at 37°C fol- lowed by 3 washes in PBS. Secondary antibody (α-rabbit IgG-alkaline phosphatase, Sigma, The Woodlands, TX) was added (1:500), incubated 1 hour at 37°C, washed 3 X in PBS and developed using a alkaline phosphatase sub- strate kit (Vector, Burlingame, CA). Viral RNA Isolation accompanied by RT-PCR Amplification of aMPV/C/US/Co N1 and N2 ORF nucleotide sequences Total RNA was isolated [38] from aMPV/C/US/Co- infected Vero cell lysates using Qiagen's "RNeasy" kit (Qiagne, Valencia, CA) according to the manufacturer's protocol. RNA was analyzed for purity by agarose gel elec- trophoresis in a 1.5% agarose gel, at 125 volts, and stained with 10 µg/ml of ethidium bromide (Sigma, The Wood- lands, TX). The aMPV N1 and N2 ORFs were reverse tran- scribed using either the N1 (5'- GAAATGTCTCTTCAGGGGATTCAG-3') and N1185C (5'- AATCATTCTGGCCTTCCTCAT-3') primer pair or the N212 (5'-ATGCAGTACGTGAGCACC-3') and N1185C (5'-AATCATTCTGGCCTTCCTCAT-3') primer pair, fol- lowed by 30 cycles of PCR [39]. RT-PCR amplification products were analyzed by agarose gel electrophoresis and the full length N1 ORF product and the N2 ORF product were excised and purified before cloning into the expres- sion vector pCR3.1-Topo (Invitrogen, Carlsbad, CA). Molecular cloning, nucleotide sequencing, and eukaryotic expression of pCR3.1-N1ORF and pCR3.1-N2ORF The N1 ORF and N2 ORF fragments of aMPV/C/US/Co were cloned into the eukaryotic expression vector pCR3.1- Topo (Invitrogen, Carlsbad, CA) according to the manu- facturer's protocol. Plasmid DNA was isolated using Qia- gen's miniprep kit (Qiagen, Valencia, CA). Double stranded sequencing with Taq polymerase (Applied Bio- systems Inc.) and fluorescent labeled dideoxynucleotides was performed with an automated sequencer [40] on both amplification products to verify identity and insure that no changes in the ORFs had been made relative to the original N gene. The pCR3.1-N1ORF and pCR3.1-N2ORF vectors were transfected into Vero cells using Virology Journal 2005, 2:31 http://www.virologyj.com/content/2/1/31 Page 8 of 9 (page number not for citation purposes) lipofectamine (Invitrogen, Carlsbad, CA). Protein was induced with IPTG (Sigma, The Woodlands, TX) at 24 hours post-transfected and total proteins were harvested by scraping. An aliquot of uninduced and induced cells were lysed in 2 X Laemmli's buffer, boiled for 5 minutes and separated by SDS-PAGE on a 4–20% Criterion (Bio- rad, Hercules, CA) gradient gel, followed by electroblot- ting onto nitrocellulose as previously described. Competing Interests The author(s) declare that they have no competing interests. Authors' contributions Dr. Alvarez was a post-doctoral associate and conducted the primary experimentation following design of peptides and production of anti-sera under the direction of Dr. Seal. Dr. Alvarez initiated writing of the draft manuscript with subsequent editing and revisions by both authors. Acknowledgements This research was supported by ARS, USDA CRIS project No. 6612-32000- 015-00D-085 and U.S. Poultry & Egg Association grant no. 404 to BSS which supported synthesis of peptides and immunization for antibodies commercially. References 1. Alexander DJ: Newcastle disease, other paramyxoviruses and pneumovirus infections. In Diseases of Poultry 11th edition. Edited by: Saif YM, Barnes HJ, Glisson JR, Fadly AM, McDougald DJ, Swayne DE. Ames, IA: Iowa State Press; 2003:63-100. 2. Jones RC: Avian pneumovirus infections: questions still unanswered. Avian Pathol 1996, 25:639-648. 3. Seal BS: Matrix protein gene nucleotide and predicted amino acid sequence demonstrate that the first US avian pneumo- virus isolate is distinct from European strains. Virus Res 1998, 58:45-52. 4. Seal BS, Sellers HS, Meinersmann RJ: Fusion protein predicted amino acid sequence of the first US avian pneumovirus iso- late and lack of heterogeneity among other US isolates. Virus Res 2000, 66:139-147. 5. Seal BS: Avian pneumoviruses and emergence of a new type in the United States of America. Anim Health Res Rev 2000, 1:67-72. 6. Lwamba HC, Alvarez R, Wise MG, Yu Q, Halvorson D, Njenga MK, Seal BS: Comparison of the full-length genome sequence of Avian metapneumovirus subtype C with other paramyxoviruses. Virus Res 2005, 107:83-92. 7. Alvarez R, Jones LP, Seal BS, Kapczynski DR, Tripp RA: Serological cross-reactivity of members of the Metapneumovirus genus. Virus Res 2004, 105:67-73. 8. Alvarez R, Lwamba HM, Kapczynski DR, Njenga MK, Seal BS: Nucle- otide and predicted amino acid sequence-based analysis of the avian metapneumovirus type C cell attachment glyco- protein gene: phylogenetic analysis and molecular epidemi- ology of U.S. pneumoviruses. J Clin Microbiol 2003, 41:1730-1735. 9. Goyal SM, Lauer D, Friendshuh K, Halvorson DA: Seroprevalence of avian pneumovirus in Minnesota turkeys. Avian Dis 2003, 47:700-706. 10. Lamb RA, Kolakofsky D: Paramyxoviridae: The Viruses and Their Replication. In Fields Virology 4th edition. Edited by: Knipe DM, and Howley PM. New York: Lippincott Williams & Wilkins; 2001:1305-1340. 11. van den Hoogen BG, de Jong JC, Groen J, Kuiken T, de Groot R, Fouchier RA, Osterhaus AD: A newly discovered human pneu- movirus isolated from young children with respiratory tract disease. Nat Med 2001, 7:719-724. 12. Steward M, Vipond IB, Millar NS, Emmerson PT: RNA editing in Newcastle disease virus. J Gen Virol 1993, 74:2539-2547. 13. Hausmann S, Jacques JP, Kolakofsky D: Paramyxovirus RNA edit- ing and the requirement for hexamer genome length. RNA 1996, 2:1033-1045. 14. Kolakofsky D, Pelet T, Garcin D, Hausmann S, Curran J, Roux L: Par- amyxovirus RNA synthesis and the requirement for hex- amer genome length: the rule of six revisited. J Virol 1998, 72:891-899. 15. Briedis DJ, Lamb RA, Choppin PW: Influenza B virus RNA seg- ment 8 codes for two nonstructural proteins. Virology 1981, 112:417-425. 16. Inglis SC, Brown CM: Spliced and unspliced RNAs encoded by virion RNA segment 7 of influenza virus. Nucleic Acids Res 1981, 9:2727-2740. 17. Bellini WJ, Englund G, Rozenblatt S, Arnheiter H, Richardson CD: Measles virus P gene codes for two proteins. J Virol 1985, 53:908-919. 18. Ahmadian G, Chambers P, Easton AJ: Detection and characteri- zation of proteins encoded by the second ORF of the M2 gene of pneumoviruses. J Gen Virol 1999, 80:2011-2016. 19. Alvarez R, Njenga MK, Scott M, Seal BS: Development of a nucle- oprotein-based enzyme-linked immunosorbent assay using a synthetic peptide antigen for detection of avian metapneu- movirus antibodies in turkey sera. Clin Diagn Lab Immunol 2004, 11:245-249. 20. Kozak M: Initiation of translation in prokaryotes and eukaryotes. Gene 1999, 234:187-208. 21. Curran J, Kolakoofsky D: Scanning independent ribosomal initi- ation of the Sendai virus X protein. EMBO J 1988, 7:2869-2874. 22. Latorre P, Kolakofsky D, Curran J: Sendai virus Y proteins are ini- tiated by a ribosomal shunt. Mol Cell Biol 1998, 18:5021-5031. 23. Dillon PJ, Gupta KC: Expression of five proteins from the Sendai virus P/C mRNA in infected cells. J Virol 1989, 63:974-977. 24. Curran J: A role for the Sendai virus P protein trimer in RNA synthesis. J Virol 1998, 72:4274-4280. 25. Vidal S, Curran J, Kolakofsky D: Editing of the Sendai virus P/C mRNA by G insertion occurs during mRNA synthesis via a virus-encoded activity. J Virol 1990, 64:239-246. 26. Locke DP, Sellers HS, Crawford JM, Schultz-Cherry S, King DJ, Mein- ersmann RJ, Seal BS: Newcastle disease virus phosphoprotein gene analysis and transcriptional editing in avian cells. Virus Res 2000, 69(9):955-68. 27. Peeters B, Verbruggen P, Nelissen F, de Leeuw O: The P gene of Newcastle disease virus does not encode an accessory X protein. J Gen Virol 2004, 85:2375-2378. 28. Collins PL, Wertz GW: The envelope-associated 22 K protein of human respiratory syncytial virus: nucleotide sequence of the mRNA and a related polytranscript. J Virol 1985, 54:65-71. 29. Ling R, Easton AJ, Pringle CR: Sequence analysis of the 22 K, SH and G genes of turkey rhinotracheitis virus and their inter- genic regions reveals a gene order different from that of other pneumoviruses. J Gen Virol 1992, 73:1709-1715. 30. Barr J, P Chambers P, Harriott P, Pringle CR, Easton AJ: Sequence of the phosphoprotein gene of pneumonia virus of mice: expression of multiple proteins from two overlapping read- ing frames. J Virol 1994, 68:5330-5334. 31. Garcia J., Garcia-Barreno B, Vivo A, Melero JA: Cytoplasmic inclu- sions of respiratory syncytial virus-infected cells: formation of inclusion bodies in transfected cells that coexpress the nucleoprotein, the phosphoprotein, and the 22 K protein. Virology 1993, 195:243-247. 32. Khattar SK, Yunus AS, Collins PL, Samal SK: Mutational analysis of the bovine respiratory syncytial virus nucleoprotein protein using a minigenome system: mutations that affect encapsi- dation, RNA synthesis, and interaction with the phosphoprotein. Virology 2000, 270:215-228. 33. Ryan KW, Portner A, Murti KG: Antibodies to paramyxovirus nucleoproteins define regions important for immunogenic- ity and nucleoprotein assembly. Virology 1993, 193:376-384. 34. Curran J, Boeck R, Lin-Marq N, Lupas A, Kolakofsky D: Paramyxo- virus phosphoproteins form homotrimers as determined by an epitope dilution assay, via predicted coiled coils. Virology 1995, 214:139-149. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Virology Journal 2005, 2:31 http://www.virologyj.com/content/2/1/31 Page 9 of 9 (page number not for citation purposes) 35. Geysen HM, Rodda SJ, Mason TJ, Tribbick G, Schoofs PG: Strategies for epitope analysis using peptide synthesis. J Immunol Methods 1987, 102:259-274. 36. Hames BD: An introduction to polyacrylamide gel electro- phoresis. In Gel Electrophoresis of Proteins: A Practical Approach Edited by: Hames BD, Rickwood D. Oxford: IRL Press; 1981:1-91. 37. Gershoni JM: Protein blotting: a manual. Methods Biochem Anal 1988, 33:1-58. 38. Chomcynski P, Sacchi N: Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987, 162:156-159. 39. Lewis JG, Chang G-J, Lanciotti RS, Trent DW: Direct sequencing of large flavivirus PCR products for analysis of genome vari- ation and molecular epidemiological investigations. J Virol Meth 1992, 38:11-24. 40. Smith LM, Sanders JZ, Kaiser RJ, Hughs P, Dodd C, Connell CR, Heines C, Kent SBH, Hood LE: Fluorescence detection in auto- mated DNA sequence analysis. Nature 1986, 321:674-679. . GTTGGGTGGG CTGATGATGT CGAAAGGACT ACAAGAGAAG CAATGGGAGC AATGG TTA GGGAAAAAGT GCAACTCA 443 aMPV/C/US/Co ACCAATACCA CAAAATCAAA GACCATCATC CCCGGATGCT CCTATCATAC TACTCTGCAT AGGAGCATTA ATCTTCACGA AGCTGGCATC. TCACATGCAG CTGAAGTAGG AATGCAGTAC GTGAGCACCA CACTGGGAGC AGAGCGTACA CAGCAGATAC TAAAGAACTC 270 aMPV /A/ UK/3b CACCGACTAT GAGCCTGCAG CAGAGGTAGG CATGCAGTAC ATTAGTACTG CTCTAGGAGC TGATAGAACT CAACAAATAC. CTTTCGGCAG AGGGTTCTGT CAGAAAGCGA GAGGTGCT AAACATTCAC 358 aMPV/C/US/Co CATACATGGG GTTGAAAGAA GT TGG-AT TGAAGAAGTT GACAAAGAGG CAAGGAAAAC CATGGCCTCA GCTACAAAGG ACAACTCAGG 444 aMPV /A/ UK/3b GATGTA-GGT